There is increasing recognition that failure of vascular grafts is a major medical problem. Particularly problematic have been saphenous vein grafts (SVG) used for coronary and peripheral arterial bypass surgeries. SVG feature obstructions related to thrombosis, neointimal hyperplasia and accelerated atherosclerosis that compromise graft patency. Patients receiving such grafts are at risk for profound medical problems including angina, myocardial infraction, the need for repeated percutaneous and surgical reveascularization procedures, and even death. See generally Motwani, J. G. and Topol, E. J. (1998) in Cardiology 97: 916; and Cameron, A. et al. (1996) in N. Engl. J. Med. 334: 216.
There is general recognition that failure of vascular grafts can be divided into two categories ie., early and late. Early graft failure (occurring within the first few months after implantation) is typically due to occlusive thrombosis. Late graft failure (occurring months to years after implantation) is due to neointimal hyperplasia and the development of accelerated atherosclerosis.
Thrombomodulin (TM) has been reported to be an important anticoagulant molecule expressed in abundance by the endothelial cell lining of all blood vessels. TM has been reported to reduce or block thrombosis due to its ability to mediate the activation of the circulating anticoagulant molecule, protein C. In a normal blood vessel, the small amount of thrombin that may be generated binds to TM expressed on endothelial cells. Binding of thrombin to TM converts thrombin's active site specificity such that it is no longer able to cleave fibrinogen to fibrin or activate cell-associated thrombin receptors. Thrombin bound to TM is, however, able to cleave protein C, thus generating activated protein C (APC). APC is a potent anticoagulant, which inhibits further thrombin generation by degrading several blood coagulation factors See eg., Jackman, R., et al., (1986) Proc. Natl. Acad Sci. U.S.A. 83:8834 and (1987) 84:6425.
There have been suggestions in the field that the level of TM expression and ability to activate protein C are important determinants of vascular thromboresistance. See eg., Mitchell C A et al (1986) Thromb Haemost. (1986) 56: 151; and Esmon, C T (1989) J. Biol. Chem. 264: 4743. Intravascular thrombosis occurs when the degree of local thrombin activation overwhelms the anticoagulant effects of the TM/APC pathway. Inherited or acquired defects in either TM or APC levels and/or function have been associated with pathologic thrombosis in humans. See Esmon C T (1989) J. Biol. Chem. 264: 4743 and references cited therein.
Denudation of the endothelial lining of arteries, as occurs during coronary or peripheral balloon angioplasty, is known to frequently result in occlusive thrombus formation as a result of enhanced local thrombin activation. Loss of TM activity and subsequent inability to generate APC consequent to endothelial cell denudation facilitates this local thrombin activation and contributes to vascular thrombus formation.
The endothelium of vein grafts implanted into the arterial circulation remains intact but appears to suffer a more subtle and chronic form of injury compared to that occurring during balloon injury of an artery. Although acute thrombosis occurs in up to 8 to 12% of SVG within the first month after implantation, little is known about the pathologic processes occurring to the endothelium that contribute to this. Specifically, little is known about changes in the expression or function of molecules that contribute to the thromboresistance of a normal blood vessel.
The neointima is understood to be a specific vessel layer that stereotypically develops after many forms of vascular injury. It is formed by proliferation and migration of smooth muscle cells out of the medial layer toward the vessel lumen. If unchecked, the neointima can encroach upon the lumen of the vessel resulting in the restriction of blood flow. In addition to causing vascular graft occlusion per se, the neointima is an avid substrate for the development of accelerated atherosclerosis.
Accelerated atherosclerosis is the major cause of late vein graft failure. Atherosclerotic plaques that develop in vascular grafts are prone to lumenal encroachment in identical fashion to plaques that develop in native arteries. Rupture of an atherosclerotic plaque in a coronary SVG can cause angina, myocardial infarction and death. Rupture of a plaque in a peripheral SVG can cause claudication and acute limb ischemia. In both cases, surgical or percutaneous procedures aimed at restoring graft patency often entails substantial risk to the patient and is frequently unsuccessful.
While the prevention of vascular graft failure is a worthwhile goal, few therapeutic options currently exist. (See Motwani J. and E. J. Topol (1998) Circulation 97: 916 and O. N. Nwasokwafor (1995) Ann Intern Med 123:528 for comprehensive reviews.) Given the prominent role of thrombosis, there have been some attempts to prevent vascular graft failure using systemic administration of antithrombotic or antiplatelet agents agents. Evidence suggests that use of such agents may marginally improve the survival of SVG, though they place the patient at risk for untoward bleeding risks. A more localized form of therapy that aggressively alters the prothrombotic phenotype of vascular grafts without concomitant adverse systemic effects is highly desirable.
Gene therapy methods, in particular, are increasingly recognized as a powerful tool for targeting the expression of pharmacologically active proteins to the vasculature. See eg., Schulick A H, et al. (1993) Circ. Res. 77: 475; Nabel E. G et al. (1990) Science 249: 1285; and Rafield, L. Et al. WO 92/07573.
See also Lim et al. (1991) Circulation 83: 2007; Flugelman et al., (1992) Circulation 85: 1110; Leclerc et al. (1992) J. Clin. Invest. 90: 936; Chapman et al. Circ. Res. 71: 27; Riessen et al. (1993) Hum. Gene Ther. 4: 749; Takeshita et al. J. Clin. Invest. (1994) 93: 652; Ohno T et al. (1994) Science 265: 781; von der Leyen, H. E. et al. PNAS (USA) (1995) 92: 1137.
Transfer of anti-sense nucleic acids that inhibit the expression of specific genes into arteries has also been reported. See Takeshita, S. et al. (1996) Laboratory Invest. 75: 487; and U.S. Pat. No. 5,652,225.
Viral mediated gene transfer is one preferred means of expressing anticoagulant molecules at the site of in vivo vascular injury. Results with first-generation replication defective adenovirus vectors have demonstrated that expression of the potent thrombin inhibitor, hirudin, can reduce thrombin-induced neointima formation following arterial injury without adverse systemic anticoagulant effects. See J. J. Rade et al. (1996) Nature Medicine 2: 293, for example.
In similar fashion, adenovirus-mediated gene transfer of TM to balloon injured arteries has been demonstrated to reduce both thrombus and neointima formation (J. M. Waugh et al. (1999) Circ Res 84:84 and (2000) Circulation 102:332). In this instance, balloon injury is expected to denude the arterial endothelium, the sole source of endogenous TM. It is not unexpected, therefore, that restoration of TM expression would decrease local thrombus formation and thrombin-induced neointima formation as was shown with adenovirus-mediated expression of hirudin (Rade, vida supra).